Novel inducible genes from alfalfa and methods of use thereof

ABSTRACT

The present invention relates to novel inducible genes from alfalfa. These genes are highly induced following mechanical harvesting or wounding of alfalfa. The genes are useful for inducible production of heterologous proteins following harvesting.

The present invention relates to recombinant protein production inplants. More particularly, the present invention relates to novelinducible genes that are expressed upon harvest, methods for isolatingsuch genes, and methods for using these genes or components therefrom.

BACKGROUND OF THE INVENTION

The mass production of recombinant molecules of commercial value is atechnical area of increasing complexity and interest. Many differentorganisms have been considered as hosts for foreign protein expressionincluding single-cell organisms such as bacteria and yeast, cellcultures of animals, fungi and plants, and whole organisms such asplants, insects, fungi and transgenic animals. In general, eachparticular organism has unique characteristics that may offer advantagesfor production of specific proteins of interest. Alternatively thespecificity of certain protein production platforms may limit utilityfor widespread applications. Thus, numerous molecular farming systemshave been developed as a means to produce proteins of commercialinterest.

Of particular interest to the subject matter of the present invention isthe expression of heterologous proteins in plant cells. Numerous foreignproteins have been expressed in whole plants and selected plant organs.Plants can offer a highly effective and economical means to producerecombinant proteins as they can be grown on a large scale with modestcost inputs and most commercially important species can now betransformed.

In order to optimize protein production and recovery, a number offactors need to be considered. These include the levels of recombinantprotein production, the temporal aspects of recombinant proteinproduction, and the stability of the final product within the plantcell. The level of protein production must be sufficient to allowaccumulation of the product in quantities that are commercially valuableand can be conveniently isolated. In many instances, it may be desiredthat the temporal expression of the product coincide with the periodwhen the crop is harvested or collected. In addition, it may be requiredthat the protein stably accumulate to appreciable levels, or be inducedto quickly accumulate to appreciable levels if the product isintrinsically unstable.

The production of heterologous proteins in plants has been achievedusing a variety of approaches. U.S. Pat. No. 6,650,307, U.S. Pat. No.5,716,802, U.S. Pat. No. 5,763,748 disclose recombinant proteinproduction using transcriptional fusions to a constitutive plantpromoter. Production of heterologous proteins in seed (U.S. Pat. No.5,504,200; U.S. Pat. No. 5,530,194; U.S. Pat. No. 6,905,186; U.S. Pat.No. 5,792,922; U.S. Pat. No. 5,948,682), fruit (U.S. Pat. No. 6,783,394;U.S. Pat. No. 4,943,674) or storage organs such as tubers (U.S. Pat. No.5,436,393, U.S. Pat. No. 5,723,757) have also been described.

A disadvantage of constitutive expression systems is that constitutiveexpression of a protein may lead to toxic effects with regards to plantgrowth. Furthermore, it is difficult to predict what interactions aforeign protein may have with other plant proteins, such as enzymes orreceptors, plant membranes, such as those of the endoplasmic reticulum,Golgi apparatus, vacuole and plasmalemma, or the host of other moleculescritical to the growth and development of the plant. Another potentialdisadvantage of a constitutive or non-inducible promoter is themetabolic cost of synthesizing the transgenic protein in all tissues atall stages of growth. If the only tissue to be harvested is the leaves,for example, it is inefficient and wasteful for the plant to produce theforeign protein in other tissues. Alternatively, if the transgeneencoded protein is labile or unstable, then production of the protein,constitutively, throughout the growth of the plant is inefficient.

Inducible systems allow the expression of an introduced gene to takeplace at a desired time in the development of a plant, under specificcircumstances or in specific tissues. For example, leaf-specificpromoters or promoters induced in the leaves by some treatment wouldrestrict synthesis to only the harvested tissue. In addition, an inducedforeign gene is potentially less likely to undergo gene silencing than atransgene controlled by a constitutive or tissue specific promoter.Furthermore, inducible transgene systems offer a method of biologicalcontainment since the foreign protein is not present in the crop untilthe application of the inducing treatment, at which time the crop isharvested. Containment of a protein produced from a foreign gene is as,or more, important than containment of the gene as the protein is thebiologically active component.

Gene expression in response to plant wounding is another potentialsource of an inducible system. U.S. Pat. No. 5,689,056, U.S. Pat. No.5,670,349, U.S. Pat. No. 5,929,304, and U.S. Pat. No. 5,777,200 disclosethe use of regulatory elements from wound inducible genes for theinduction of heterologous protein synthesis in plants. However, thevalue of these wound-inducible promoters may be limited since woundingof the plant also induce other genes, such as proteases, that cannegatively impact the production of the recombinant protein. It is alsonot clear that these regulatory elements provide sufficient levels ofexpression to cause accumulation of the recombinant protein tosubstantial levels, especially when the response is localized to thesite of wounding (e.g. HMG2 promoter, U.S. Pat. No. 5,689,056). Althoughthe expression levels with such promoters can be enhanced by applyingmore extensive wounding treatments or chemical inducers such as methyljasmonate, this entails additional costs.

Thus, although promoters involved in inducible systems can providepowerful tools for control of transgenes in plants, many obstacles arefaced in utilizing these regulatory elements. Inducible promoter systemsmust enable the precise timing and location of expression of suchtransgenes in order to be commercially useful. In this regard,regulatory elements that can be induced under precise conditionsamenable to cultivation practices are desired. More particularly, thereis a need for regulatory elements that are induced, specifically, duringharvesting conditions.

Volenec et al. (“Molecular analysis of alfalfa root vegetative storageproteins” pp59-73 in Molecular and Cellular Technologies for ForageImprovement, CSSA Spec Publ. No. 26, 1998) have characterized thechanges that ensue in root tissue following harvest and shoot regrowthof alfalfa (Medicago sativa L.). However, no specific regulatoryelements were identified or characterized in any manner.

Ferullo et al (Crop. Sci. 1996 36, 1011-1016) disclose proteins that arespecific to harvesting conditions of alfalfa. However the structure orfunction of these proteins was not characterized and is unknown;moreover, there is no indication of the nature of the genes expressed inharvested shoot tissue of alfalfa during harvesting. Furthermore, thereis no suggestion as to the use of regulatory elements associated withthese genes for induction of heterologous gene expression in plants in aharvest-inducible manner.

Coupe et al. (WO 00/31251) disclose the characterization of a promoterfrom asparagine synthetase and its use in post harvest gene expression.

It is an object of the invention to overcome disadvantages of the priorart.

The above object is met by the combinations of features of the mainclaims, the sub-claims disclose further advantageous embodiments of theinvention.

SUMMARY OF THE INVENTION

The present invention relates to recombinant protein production inplants. More particularly, the present invention relates to novelinducible genes that are expressed upon harvest, methods for isolatingsuch genes, as well as methods for using these genes.

The present invention provides a method (A) for isolating aharvest-inducible DNA sequence comprising:

i) constructing one or more first cDNA libraries comprising cDNAsequences expressed in harvested tissue;

ii) preparing one or more second cDNA libraries comprising cDNAsequences expressed in tissues of an intact plant prior to harvest; and

iii) identifying harvest-inducible cDNA sequences. The expression of theharvest-inducible cDNA sequences may be analyzed to determineinducibility of the harvest-inducible cDNA sequences upon harvesting.

An example of identifying harvest-induced cDNA sequences (step iii))include subtractive hybridization of the first cDNA library with anexcess of the second cDNA library, however, other methods may also beused as known in the art.

The present invention also relates to an isolated harvest-inducible cDNAsequence obtained according to the above method (A).

The present invention embraces an isolated harvest-inducible cDNAsequence selected from the group consisting of:

i) SEQ ID NO:1, a complement thereof, a fragment of SEQ ID NO:1, acomplement of a fragment of SEQ ID NO:1, a nucleic acid that hybridizesto SEQ ID NO:1 under stringent hybridization conditions, a nucleic acidthat hybridizes to a complement of SEQ ID NO:1 under stringenthybridization conditions, a nucleic acid that hybridizes to a fragmentof SEQ ID NO:1 under stringent hybridization conditions, or a nucleicacid that hybridizes to a complement of fragment of SEQ ID NO:1 understringent hybridization conditions;

ii) SEQ ID NO:2, a complement thereof, a fragment of SEQ ID NO:2, acomplement of a fragment of SEQ ID NO:2, a nucleic acid that hybridizesto SEQ ID NO:2 under stringent hybridization conditions, a nucleic acidthat hybridizes to a complement of SEQ ID NO:2 under stringenthybridization conditions, a nucleic acid that hybridizes to a fragmentof SEQ ID NO:2 under stringent hybridization conditions, or a nucleicacid that hybridizes to a complement of fragment of SEQ ID NO:2 understringent hybridization conditions; and

iii) SEQ ID NO:3, a complement thereof, a fragment of SEQ ID NO:3, acomplement of a fragment of SEQ ID NO:3, a nucleic acid that hybridizesto SEQ ID NO:3 under stringent hybridization conditions, a nucleic acidthat hybridizes to a complement of SEQ ID NO:3 under stringenthybridization conditions, a nucleic acid that hybridizes to a fragmentof SEQ ID NO:3 under stringent hybridization conditions, or a nucleicacid that hybridizes to a complement of fragment of SEQ ID NO:3 understringent hybridization conditions, the stringent hybridizationconditions comprising, hybridization overnight (12-24 hrs) at 42° C. inthe presence of 50% formamide, followed by washing, or 5×SSC at about65° C. for about 12 to about 24 hours, followed by washing in 0.1×SSC at65° C. for about one hour.

Also provided in this invention is a method (B) for isolating a harvestinducible regulatory element comprising,

i) identifying genomic DNA sequences 3′ and 5′ corresponding to theharvest-inducible cDNA identified using method (A); and

ii) analyzing the genomic DNA, and identifying the harvest-inducibleregulatory element.

This method (B) may further comprise a step of:

iii) testing the harvest-inducible regulatory region within a transgenicplant or plant cell.

The present invention also provides a harvest-inducible regulatoryelement obtained using the method (B).

The present invention also pertains to a harvest-inducible regulatoryelement selected from the group consisting of:

i) SEQ ID NO:4, a complement thereof, a fragment of SEQ ID NO:4, acomplement of a fragment of SEQ ID NO:4, a nucleic acid that hybridizesto SEQ ID NO:4 under stringent hybridization conditions, a nucleic acidthat hybridizes to a complement of SEQ ID NO:4 under stringenthybridization conditions, a nucleic acid that hybridizes to a fragmentof SEQ ID NO:4 under stringent hybridization conditions, or a nucleicacid that hybridizes to a complement of fragment of SEQ ID NO:4 understringent hybridization conditions;

ii) SEQ ID NO:5, a complement thereof, a fragment of SEQ ID NO:5, acomplement of a fragment of SEQ ID NO:5, a nucleic acid that hybridizesto SEQ ID NO:5 under stringent hybridization conditions, a nucleic acidthat hybridizes to a complement of SEQ ID NO:5 under stringenthybridization conditions, a nucleic acid that hybridizes to a fragmentof SEQ ID NO:5 under stringent hybridization conditions, or a nucleicacid that hybridizes to a complement of fragment of SEQ ID NO:5 understringent hybridization conditions; and

iii) SEQ ID NO:6, a complement thereof, a fragment of SEQ ID NO:6, acomplement of a fragment of SEQ ID NO:6, a nucleic acid that hybridizesto SEQ ID NO:6 under stringent hybridization conditions, a nucleic acidthat hybridizes to a complement of SEQ ID NO:6 under stringenthybridization conditions, a nucleic acid that hybridizes to a fragmentof SEQ ID NO:6 under stringent hybridization conditions, or a nucleicacid that hybridizes to a complement of fragment of SEQ ID NO:6 understringent hybridization conditions,

the stringent hybridization conditions comprising, hybridizationovernight (12-24 hrs) at 42° C. in the presence of 50% formamide,followed by washing, or 5×SSC at about 65° C. for about 12 to about 24hours, followed by washing in 0.1×SSC at 65° C. for about one hour,wherein the regulatory element exhibits harvest-inducible activity.

Also provided in the present invention is a construct comprising theharvest-inducible regulatory element as just defined, operably linkedwith a heterologous nucleotide sequence of interest and a terminatorregion. The present invention also embraces a vector comprising the DNAconstruct as just defined. Furthermore, this invention pertains to aplant, plant tissue, plant seed, plant cell, or progeny therefrom,comprising the construct as just defined.

The present invention relates to a construct comprising a heterologousnucleotide sequence operably linked to said harvest-inducible regulatoryelement defined above, where the harvest-inducible regulatory elementfurther comprises a nucleotide sequence encoding a harvest-inducibleprotein or fragment thereof. The present invention also embraces avector comprising the DNA construct as just defined. Furthermore, thisinvention pertains to a plant, plant tissue, plant seed, plant cell, orprogeny therefrom, comprising the construct as just defined

The present invention also provides a method (C) for production of aheterologous protein into a plant comprising:

i) introducing a construct comprising a harvest-inducible regulatoryelement operably linked with a heterologous nucleotide sequence ofinterest and a terminator region, to the plant to obtain a transformedplant, where the harvest-inducible regulatory element is selected fromthe group consisting of:

SEQ ID NO:4, or a fragment thereof;

SEQ ID NO:5, or a fragment thereof;

SEQ ID NO:6, of a fragment thereof;

a nucleic acid that hybridizes to SEQ ID NO:4, 5, 6, or a complement ofSEQ ID NO:4, 5, 6 under stringent hybridization conditions; and

a nucleic acid that hybridizes to a fragment of SEQ ID NO:4, 5, 6, or acomplement of SEQ ID NO:4, 5, 6 under stringent hybridizationconditions, the stringent hybridization conditions comprising,hybridization overnight (12-24 hrs) at 42° C. in the presence of 50%formamide, followed by washing, or 5×SSC at about 65° C. for about 12 toabout 24 hours, followed by washing in 0.1×SSC at 65° C. for about onehour;

ii) growing the transformed plant; and

iii) harvesting the transformed plant thereby inducing expression of theheterologous protein.

The step of harvesting (step iii) may be followed by:

iv) isolating the heterologous protein from the transformed plant.

Furthermore, the step of isolating (step iv)) may be followed by a stepof purification of the heterologous protein.

The present invention also pertains to a method (D) for production of aheterologous protein comprising,

i) providing a plant transformed with a construct comprising aharvest-inducible regulatory element operably linked with a heterologousnucleotide sequence of interest and a terminator region, where theharvest-inducible regulatory element is selected from the groupconsisting of

SEQ ID NO:4, or a fragment thereof;

SEQ ID NO:5, or a fragment thereof;

SEQ ID NO:6, of a fragment thereof;

a nucleic acid that hybridizes to SEQ ID NO:4, 5, 6, or a complement ofSEQ ID NO:4, 5, 6 under stringent hybridization conditions; and

a nucleic acid that hybridizes to a fragment of SEQ ID NO:4, 5, 6, or acomplement of SEQ ID NO:4, 5, 6 under stringent hybridizationconditions,

the stringent hybridization conditions comprising, hybridizationovernight (12-24 hrs) at 42° C. in the presence of 50% formamide,followed by washing, or 5×SSC at about 65° C. for about 12 to about 24hours, followed by washing in 0.1×SSC at 65° C. for about one hour, andthe harvest-inducible regulatory element further comprises a nucleotidesequence encoding a harvest-inducible protein or fragment thereof;

ii) growing the transformed plant; and

iii) harvesting the transformed plant to induce expression of theheterologous protein.

The step of harvesting (step iii) may be followed by:

iv) isolating the heterologous protein from the transformed plant.

Furthermore, the step of isolating (step iv)) may be followed by a stepof purification of the heterologous protein.

The harvest-inducible regulatory elements can be used to control theexpression of a heterologous DNA sequence, such that the heterologousDNA sequence is only expressed in response to harvesting, thus providinga convenient system for the production of novel proteins. Accordingly,another aspect of the present invention is directed to DNA constructscomprising a harvest-inducible regulatory element operably linked with aheterologous nucleotide sequence of interest and a terminator region.

In order to enhance translation, stability or recovery of theheterologous or foreign protein, the nucleotide sequence encoding theheterologous protein can be operably linked to a harvest-inducible geneencoding a portion of a harvest-inducible protein and its correspondingharvest-inducible regulatory element. Accordingly, another aspect of thepresent invention relates to a DNA construct comprising a heterologousnucleotide sequence encoding a heterologous protein of interest operablylinked to an isolated harvest inducible regulatory element and a portionof the harvest-inducible gene encoding a harvest-inducible protein orfragment thereof.

The DNA constructs may be ligated or incorporated into an appropriatevector and used to transform plants in order to express heterologousproteins in plants. Accordingly, another aspect of the invention isdirected to a plant, plant tissue, plant seed, or plant cell comprisinga harvest-inducible regulatory element operably linked with ahetorologous nucleotide sequence and a terminator region.

In yet another aspect of the invention, transgenic plants are produced,the plants comprising a harvest-inducible transgene, the transgenecomprising a harvest-inducible regulatory element operably linked to aheterologous nucleotide sequence and a terminator region. The transgenemay encode a protein of veterinary or pharmaceutical or biologicalactivity, where the activity is useful for administration to livestockby feeding of whole or parts of harvested plant.

This summary of the invention does not necessarily describe allnecessary features of the invention but that the invention may alsoreside in a sub-combination of the described features.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1 shows a schematic diagram of PCR-Select cDNA substraction librarymethod for isolating harvest-inducible cDNA clones (see Example 1 formore detail of the method). cDNA containing harvest-specific transcriptsis referred to as the “tester” cDNA and the cDNA from the non-harvestedplants, referred to as “driver.” cDNA Type “e” molecules are formed onlyif the sequence is up-regulated in the tester cDNA. Solid linesrepresent the Rsa I digested tester or driver cDNA. Solid boxesrepresent the outer part of the Adaptor 1 and 2R longer strands andcorresponding PCR primer 1 sequence. Clear boxes represent the innerpart of Adaptor 1 and the corresponding nested PCR primer 1 sequence;shaded boxes represent the inner part of Adaptor 2R and thecorresponding nested PCR primer 2R sequence

FIG. 2 shows sequences of PCR-Select cDNA synthesis primer (SEQ IDNO:12), adaptors 1 and 2R (SEQ ID Nos: 13-14), PCR primer 1 (SEQ ID NO:15) and nested PCR primers 1 and 2R (SEQ ID Nos: 16-17). When theadaptors are ligated to Rsa1-digested cDNA, the Rsa1site is restored.

FIG. 3 shows Northern blot analysis of the expression of cDNA H7following harvest of leaf tissue. RNA was isolated from alfalfa leavesand probed with H7 (SEQ ID NO:1). Leaves obtained before harvest (lanes1, 5), 45 min post harvest (lanes 2, 6), 6 hours post harvest (lanes 3,7) and 24 hours post harvest (lanes 4, 8). H7 RNA is not detected inalfalfa leaves in non-harvest, i.e. pre-harvest conditions nor followingwounding or heat treatments (data not shown).

FIG. 4 shows Northern blot analysis of cDNA H11(SEQ ID NO:2) underharvesting and heat shock conditions of treatment of alfalfa leaves. RNAextracted from leaves of: lane 1, non-treated plants; lanes 2-5, plantsin harvested conditions for 30 min, 2 hours, 6 hours, 24 hours; lanes 6and 7, plants subjected to 15 or 30 min of heat shock at 38° C. RNA wasprobed with H11 cDNA clone (SEQ ID NO:2). Arrow indicates majortranscript of H11.

FIG. 5 shows Northern blot analysis of cDNA H11 (SEQ ID NO:2) followingwounding of alfalfa leaves. RNA extracted from: lane 1, non-woundedplants; lanes 2-4, plants were wounded using a scalpel and RNA extractedafter 45 min, 6, hours, and 24 hours post wounding. RNA was probed withH11 cDNA clone (SEQ ID NO:2)

FIG. 6 shows a Northern blot analysis of cDNA clone H12 (SEQ ID NO:3)following harvesting and heat shock treatments of alfalfa leaves. RNAextracted from leaves of: lane 1, non-treated plants; lanes 2-5, plantsin harvested conditions for 30 min, 2 hours, 6 hours and 24 hours; lanes6 and 7, plants subjected to 15 or 30 min of heat shock at 38° C.

FIG. 7 shows a diagram of a vector construct containing the putativepromoter region of an H1 gene and a GFP reporter gene. The arrowsrepresent the left and right borders of the T-DNA region of a binaryvector used in Agrobactrium-mediated gene transfer. P represents apromoter used to drive a selective marker such as the resistance gene tothe antibiotic neomycin, and T represents a terminator regulatoryelement such as that derived from nopaline synthase, the CaMV 35S geneor from a plant gene such as H7. The GFP(EGF) coding region followingthe H7 promoter could represent the coding region of the fluorescentprotein, a fusion protein consisting of GFP and EGF (epidermal growthfactor), or simply the coding region of EGF alone or any other sequenceencoding a peptide or protein with medical or veterinary properties.

FIG. 8 shows the H7 genomic sequence (SEQ ID NO:7), including the 5′flanking regulatory, and coding, regions. The regulatory region is fromnucleotide 1 to nucleotide 634 (SEQ ID NO:4) which is the transcriptioninitiation site (bold, large A). Putative TATA boxes are enclosed in aboxed outline. The coding region of the H7 gene is in bold italics andbegins at nucleotide 675 and ends at nucleotide 1148 (SEQ ID NO:1); thesingle letter amino acid sequence of the protein is under the DNAsequence. The 3′ UTR starts at 1149 up to the poly A sequence.

FIG. 9 shows the H11 genomic sequence (SEQ ID NO:8) and associatedregions. Regulatory region (SEQ ID NO:5, nucleotides 1 to about 438),intron (nucleotides 651-772) and 3′ UTR (nucleotides 1239 to the polyAsequence) are in lower case; coding region (nucleotides 439-650 and773-1238; SEQ ID NO:2) is in upper case and bold.

FIG. 10 shows the H12 genomic sequence (SEQ ID NO:9) and associatedregions. Regulatory region (nucleotides 1-936; SEQ ID NO:6), 3′ UTR(nucleotides 1720-1906); coding region (nucleotides 976-1720; SEQ IDNO:3) is in upper case and bold.

FIG. 11 shows binary vectors containing harvest-inducible promotersfused to the GUS gene. Pro: promoter; T: terminator; RB/LB: right/leftborders from T-DNA region of Ti plasmid of Agrobacterium; ³⁵S: from theregulatory region of the 35S transcript of the cauliflower mosaic virus;

: catalase intron in GUS gene.

FIG. 12 shows the expression of H7-GUS in tobacco at time zero (left)and 24 hours post-harvest (right).

FIG. 13 shows GUS expression in random samples from M. truncatula plantsgrown from seedling co-cultivated with Agrobacterium. Upper left plate:H12-GUS (24 hours post-harvest); upper right plate: H11-GUS (24 hourspost-harvest); lower left: H7-GUS (24 hours post-harvest); lower middle:35S-GUS (24 hours post-harvest); lower right: untransformed control. Thechimeric nature of transformation events results in non-blue sectors ofplants, and hence leaves and stems showing no blue coloration.

DESCRIPTION OF PREFERRED EMBODIMENT

The present invention relates to recombinant protein production inplants. More particularly, the present invention relates to novelinducible genes that are expressed upon harvest, and methods to usethese genes.

The following description is of a preferred embodiment by way of exampleonly and without limitation to the combination of features necessary forcarrying the invention into effect.

The singular forms “a,” “an” and “the” include plural reference unlessthe context clearly dictates otherwise.

Two DNA sequences are “operably linked” if the nature of the linkagedoes not interfere with the ability of the sequences to effect theirnormal functions relative to each other. For instance, a promoter, or aregulatory region would be operably linked to a coding sequence if thepromoter or regulatory region were capable of effecting transcription ofthat coding sequence.

By “regulatory region” or “regulatory element” it is meant a nucleicacid sequence that has the property of controlling the expression of aDNA sequence that is operably linked with the regulatory region. Suchregulatory regions may include promoter or enhancer regions, and otherregulatory elements recognized by one of skill in the art. By “promoter”it is meant the nucleotide sequences at the 5′ end of a coding region,or fragment thereof that contain all the signals essential for theinitiation of transcription and for the regulation of the rate oftranscription.

The term “gene” is used in accordance with its usual definition in theart to mean an operatively linked group of nucleic acid sequences. Byoperatively linked it is meant that the particular sequences interacteither directly or indirectly to carry out their intended function, suchas mediation or modulation of gene expression. The interaction ofoperatively linked sequences may for example be mediated by proteinsthat in turn interact with the sequences. A transcriptional regulatoryregion and a sequence of interest are operably linked when the sequencesare functionally connected so as to permit transcription of the sequenceof interest to be mediated or modulated by the transcriptionalregulatory region.

By “coding sequence of interest” it is meant any coding sequence that isto be expressed in a transformed plant. Such a coding sequence ofinterest may include, but is not limited to, a coding sequence thatencodes an antigen, such as a viral coat protein or microbial cell wallor toxin proteins or various other antigenic peptides, such as swineviral antigen. Other proteins or peptides of interest include growthfactors, such as epidermal growth factor, antimicrobial peptides, suchas defensins, and other peptides with physiological and immunologicalproperties, such as opioids and cytokines, or other pharmaceuticallyactive proteins. Such proteins include, but are not limited to,interleukins, insulin, G-CSF, GM-CSF, hPG-CSF, M-CSF or combinationsthereof, interferons, for example, interferon-□α, interferon-β,interferon-τ, blood clotting factors, for example, Factor VIII, FactorIX, or tPA or combinations thereof. Furthermore, a coding sequence ofinterest may also encode an industrial enzyme, protein supplement,nutraceutical, or a value-added product for feed, food, or both feed andfood use. Examples of such proteins include, but are not limited toproteases, oxidases, phytases, chitinases, invertases, lipases,cellulases, xylanases, enzymes involved in oil biosynthesis etc. Otherprotein supplements, nutraceuticals, or a value-added products includenative or modified seed storage proteins and the like. The invention isnot limited by the source or the use of the recombinant polypeptide orheterologous nucleotide sequence encoding the polypeptide.

A “transgenic” organism, such as a transgenic plant, is an organism intowhich foreign DNA has been introduced. A “transgenic plant” encompassesall descendants, hybrids, and crosses thereof, whether reproducedsexually or asexually, and which continue to harbour the foreign DNA.

A “vector” may be any of a number of nucleic acid sequences into which adesired sequence may be inserted by restriction and ligation. A vectortypically carries its own origin of replication, one or more uniquerecognition sites for restriction endonucleases which can be used forthe insertion of foreign DNA, and usually selectable markers such asgenes coding for antibiotic resistance or herbicide resistance, andoften recognition sequences (e.g. promoter) for the expression of theinserted DNA. Common vectors include, but are not limited to, viralvectors, plasmids, phage, phagmids, and cosmids. Vectors may also bemodified to contain a region of homology to an Agrobacterium tumefaciensvector, preferably a T-DNA border region from Agrobacterium tumefaciens.Further, vectors can comprise a disarmed plant tumor inducing plasmid ofAgrobacterium tumefaciens.

Unless defined otherwise all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs.

A nucleotide sequence is said to exhibit “harvest-inducible regulatoryactivity” when the nucleotide sequence (the first nucleotide sequence,or harvest inducible regulatory element) regulates expression of asecond nucleotide sequence to which it is operably linked, followingharvesting of plant tissue. A regulatory region (the first nucleotidesequence) that exhibits harvest-inducible regulatory activity (aharvest-inducible regulatory element) may also exhibit activity underother conditions for example but not limited to, wounding, heat shock,or other environmental stresses. Harvest-inducible regulatory activitymay result in an increase in the expression of the second nucleotidesequence, or a decrease in the expression of the second nucleotidesequence, when compared to the expression of the second nucleotidesequence under non-harvest conditions. A harvest-inducible regulatoryelement may therefore be active in increasing or decreasing expressionof a second nucleotide sequence to which it is operably linked, relativeto the expression of the second nucleotide sequence under non-harvestconditions.

The present invention provides regulatory elements obtained from genesthat exhibit modified expression upon harvest of plant tissue.Furthermore, the present invention pertains to the use of theseregulatory regions for the expression of heterologous proteins inplants. The present invention is also directed to chimeric constructscontaining a DNA of interest operatively linked to a harvest-inducibleregulatory element of the present invention. Any exogenous gene, or geneof interest comprising a coding sequence of interest, can be used andmanipulated according to the present invention to result in theexpression of the exogenous gene.

Harvesting, as is typically carried out in the field involves cutting ofplants at the base of the stem at a desired stage of growth, for examplebut not limited to the late bud stage, and laying cut material in aswath followed by drying at ambient field moisture and temperatureconditions to a specific moisture level appropriate for baling orensiling.

The present invention provides a method to isolate harvest-induciblegenes comprising:

i) constructing a cDNA subtraction library using any suitable methodknown in the art, from harvested and non-harvested tissues andidentifying clones unique to the harvested tissues; and

ii) identifying sequences preferentially expressed in response toharvesting.

These harvest-inducible cDNA sequences may be characterized usingNorthern analysis and sequencing.

Examples of harvest-induced cDNA sequences that are preferentiallyexpressed in response to harvesting conditions, and generally notexpressed under other conditions typical of cultivation, include, butare not limited to H7, H11 and H12 (SEQ ID NO's: 1-3, respectively),fragments thereof, sequences that hybridize to SEQ ID NO's: 1-3,fragments thereof under stringent hybridization conditions as known inthe art, and complements of these sequences, or sequences that exhibit a80%-100% similarity using sequence alignment protocols, for example, butnot limited to, BLAST. The coding region of H7 (SEQ ID NO:1) comprisesnucleotides 675-1148 of FIG. 8. The coding region of H11 (SEQ ID NO:2)comprises nucleotide about 439-650 and nucleotides 773-1238 of FIG. 9.The coding region of H12 (SEQ ID NO:3) comprises nucleotides about976-1720 of FIG. 10.

Stringent hybridization conditions are known within the art (e.g.Sambrook et al, 1989, in “Molecular cloning: a laboratory manual”,2^(nd) edition, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory,which is incorporated herein by reference), and may comprise,hybridization overnight (12-24 hrs) at 42° C. in the presence of 50%formamide, followed by washing using standard protocols (Sambrook et al,1989), or 5×SSC at about 65° C. for about 12 to about 24 hours, followedby washing in 0.1×SSC at 65° C. for about one hour.

Sequence comparisons between two or more polynucleotides (orpolypeptides, as required) may be performed by comparing portions of thetwo sequences over a comparison window to identify and compare localregions of sequence similarity. The percentage similarity is calculatedby: (a) determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions; (b) dividing the number ofmatched positions by the total number of positions in the window ofcomparison; and, (c) multiplying the result by 100 to yield thepercentage of sequence identity. Optimal alignment of sequences forcomparison may be conducted by utilizing readily available sequencecomparison and multiple sequence alignment algorithms are, respectively,the Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al.1990. J. Mol. Biol. 215:403; Altschul, S. F. et al. 1997. Nucleic AcidsRes. 25: 3389-3402) and ClustalW programs. BLAST is available on theInternet at http://www.ncbi.nlm.nih.gov and a version of ClustalW isavailable at http://www2.ebi.ac.uk using default parameters (for examplebut not limited to, Program: blastn; Database:nr; low complexity; Expect10; Word size 11).

Using the above method, harvest-inducible cDNA's may be identified andcharacterized. For example, which is not to be considered limiting inany manner, expression of H7 or H12 is not detected in pre-harvestedplant material yet expression increases significantly after tissue isharvested (see FIGS. 3 and 6, respectively). Similarly, H11 expressionincreases significantly after harvesting (see FIG. 4). However, increasein expression of H11 is also observed in response to heat shock andwounding (FIGS. 4, 5).

Genome walking may be used to identify regulatory regions associatedwith a harvest-inducible cDNA (see Example 3). Alternatively,harvest-induced cDNA sequences may be used to isolate regulatoryelements associated with one or more genomic sequences that are similarto harvest induced cDNA sequences, or that hybridize to harvest inducedcDNA sequences under specified hybridization conditions. Regulatoryelements thus obtained are capable of conferring harvest-inducibilityupon one or more coding sequences of interest that are operably linkedto the regulatory elements.

Therefore, the present invention, also relates to the isolation ofregulatory elements comprising,

i) isolating genomic DNA from a plant; and

ii) identifying a regulatory region within the genomic DNA usingharvest-induced cDNA.

The identified regulatory region may then be further characterized bysequencing and expression analysis, for example, the regulatory regionmay be used to drive expression of a marker sequence and the activity ofthe regulatory region analyzed in various tissues and under differentenvironmental or harvest conditions. The regulatory region may beidentified using genomic walking using PCR primers identified fromharvest-inducible cDNA's. However, other methods that are known in theart may also be used.

A regulatory element identified using the above method may be operablylinked with a coding sequence of interest, for example a marker gene,see for example, but not limited to the construct of FIG. 7, and testedto demonstrate harvest inducibility using any suitable technique, forexample but not limited to biolistics, protoplast, or Agrobacteriumtransformation, as disclosed herein.

Using the above methods, one or more regulatory regions may beidentified that are capable of conferring harvest-inducibility upon acoding sequence of interst operably linked to the regulatory region.Examples, which are not to be considered limiting in any manner, ofregulatory elements obtained using the methods of the present inventioninclude SEQ ID NO's: 4-6 (regulatory regions of H7, nucleotides 1-634 ofFIG. 8; H11, nucleotides 1 to about 438 of FIG. 9; and H12, nucleotides1-935 of FIG. 10, respectively), fragments thereof, or sequences thathybridize to SEQ ID NO's: 4-6, or their complement, under stringenthybridization conditions (e.g. hybridization overnight (12-24 hrs) at42° C. in the presence of 50% formamide, followed by washing usingstandard conditions, or 5×SSC at about 65° C. for about 12 to about 24hours, followed by washing in 0.1×SSC at 65° C. for about one hour) orthat exhibit a 80%-100% similarity using sequence alignment protocols,for example, but not limited to, BLAST (Program: blastn; Database:nr;low complexity; Expect 10; Word size 11), provided the sequence exhibitsharvest-inducible regulatory element activity.

The present invention therefore provides DNA constructs useful forproducing a protein or peptide of interest within a plant. Examples ofDNA constructs of the present invention, which are not to be consideredlimiting in any manner, include a coding sequence of interest operablylinked to a harvest inducible regulatory element, or a nucleotidesequence encoding the protein of interest fused to a nucleotide sequenceencoding a harvest-induced protein, or a portion thereof, where thenucleotide sequence encoding the harvest-induced protein or portionthereof is operably linked to a harvest-inducible regulatory element.This latter construct may be used to ensure stability of a protein ofinterest following expression in a plant. It is also contemplated thatpeptide sequences that facilitate isolation, purification, or both ofthe protein of interest, for example affinity tags, protease cleavagesites, or both may be included in the DNA constructs. These DNAconstructs may be introduced into an expression cassette suitable forplant transformation.

The present invention is also directed to a method for production of aprotein or peptide of interest comprising,

i) introducing a construct comprising a coding sequence of interestoperably linked to a harvest inducible regulatory element into a plant,to obtain a transgenic plant;

ii) growing the transgenic plant; and

iii) harvesting the transgenic plant thereby inducing production of theprotein of interest.

If required, the protein or peptide of interest may be recovered afterharvest.

Additionally, the present invention provides a method for production ofa protein or peptide of interest comprising,

i) providing a plant comprising a construct comprising a coding sequenceof interest operably linked to a harvest inducible regulatory element;

ii) growing the plant; and

iii) harvesting the plant thereby inducing production of the protein orpeptide of interest.

If required, the protein or peptide of interest may be recovered afterharvest.

The HI promoters of the present invention are similarly regulated acrossplant families and genera, such that they have applications in crops ofvarious species. Thus, this method may be used with any desired plant,for example but not limited to potato, tomato, canola, corn, soybean,alfalfa, pea, lentil, other forage legumes such as clover, trefoil,forage grasses such as timothy, ryegrass, brome grass, fescue or othercereal grasses used for forage such as barley, wheat, sudan grass,sorgham.

The present invention also provides a method for enhancing translation,stability, recovery, or a combination thereof, of a protein or peptideof interest upon harvest of a plant tissue comprising:

i) introducing a construct comprising a coding sequence of interestfused to a nucleotide sequence encoding a harvest-induced protein, or aportion thereof into a plant to obtain a transgenic plant, where thenucleotide sequence encoding the harvest-induced protein or portionthereof is operably linked to a harvest-inducible regulatory element;

ii) growing the transgenic plant; and

iii) harvesting of the transgenic plant to induce expression of theprotein or peptide of interest.

If required, the protein or peptide of interest may be recovered afterharvest.

Furthermore, a method for enhancing translation, stability, recovery, ora combination thereof, of a protein or peptide of interest upon harvestof a plant tissue is also provided, the method comprising:

i) providing a plant comprising a construct, the construct comprising acoding sequence of interest fused to a nucleotide sequence encoding aharvest-induced protein, or a portion thereof, where the nucleotidesequence encoding the harvest-induced protein or portion thereof isoperably linked to a harvest-inducible regulatory element;

ii) growing the plant; and

iii) harvesting the plant to induce expression of the protein or peptideof interest.

If required, the protein or peptide of interest may be recovered afterharvest.

As the HI promoters of the present invention are similarly regulatedacross plant families and genera, this method may be used with anydesired plant, for example but not limited to potato, tomato, canola,corn, soybean, alfalfa, pea, lentil, other forage legumes such asclover, trefoil, forage grasses such as timothy, ryegrass, brome grass,fescue or other cereal grasses used for forage such as barley, wheat,sudan grass, sorgham.

With either of the above methods, the protein of interest may beisolated and purified, as required, using standard techniques known inthe art.

The methods provided herein may be used to produce heterologous proteinsof interest in a plant, and allows for the production of crop plantsspecifically designed for molecular farming wherein plants produce novelproteins with commercial or pharmaceutical applications.

Of particular interest are those proteins or peptides that may have atherapeutic value, for example vaccines. Vaccines produced by themethods of the present invention include antigens, such as viral coatproteins or microbial cell wall or toxin proteins or various otherantigenic peptides, such as swine viral antigen. Other proteins orpeptides of interest include growth factors, such as epidermal growthfactor, antimicrobial peptides, such as defensins, and other peptideswith physiological and immunological properties, such as opioids andcytokines. The invention is not limited by the source or the use of therecombinant polypeptide or heterologous nucleotide sequence encoding thepolypeptide.

Examples of other proteins which may be produced in plants or crops byusing the regulatory elements, constructs, or methods of the presentinvention, and that may be considered as genes of interest, include butare not limited to, industrial enzymes, for example, proteases,carbohydrate modifying enzymes such as alpha amylase, glucose oxidase,cellulases, hemicellulases, xylanases, mannases or pectinases, (forexample U.S. Pat. No. 5,824,870, U.S. Pat. No. 5,767,379, U.S. Pat. No.5,804,694). Additionally, the production of enzymes particularlyvaluable in the pulp and paper industry such as ligninases or xylanasesis also contemplated (for example U.S. Pat. No. 5,981,835). Otherexamples of enzymes include phosphatases, oxidoreductases and phytases(for example U.S. Pat. No. 5,714,474). The number of industriallyvaluable enzymes is large and plants can offer a convenient vehicle forthe mass production of these proteins at costs anticipated to becompetitive with fermentation, provided the production system isefficient and easily manipulated. Also contemplated are protein-basedelastomers to replace allergenic compounds such as latex.

Additionally, molecular farming is also being contemplated for use inthe production and delivery of vaccines (for example, U.S. Pat. No.6,136,320, U.S. Pat. No., 5,914,123, U.S. Pat. No. 5,679,880, U.S. Pat.No. 5,679,880, U.S. Pat. No. 5,654,184, U.S. Pat. No. 5,612,487, U.S.Pat. No. 6,034,298, WO 99/37784A1), antibodies (for example, WO97/2900A1, U.S. Pat. No. 5,959,177, U.S. Pat. No. 5,202,422, U.S. Pat.No. 5,639,947, U.S. Pat. No. 6,046,037), peptide hormones (for example,U.S. Pat. No. 5,487,991, WO 99/6740A2), blood factors and similartherapeutic molecules. It has been postulated that edible plants whichhave been engineered to produce selected therapeutic agents couldprovide a means for drug delivery which is cost effective andparticularly suited for the administration of therapeutic agents inrural or under developed countries. The plant material containing thetherapeutic agents could be cultivated and incorporated into the diet(for example U.S. Pat. No. 5,484,719). Similarly, plants used for animalfeed can be engineered to express veterinary biologics that can provideprotection against animal disease, (for example WO 99/37784A1).

The DNA sequence encoding the protein of interest may be synthetic,naturally derived, or a combination thereof. Dependent upon the natureor source of the DNA encoding the polypeptide of interest, it may bedesirable to synthesize the DNA sequence with codons that representplant-preferred codons. It is contemplated that the coding region of theprotein of interest can be joined to the coding sequence of aharvest-inducible protein obtained as described herein, to aid instability or accumulation, or to provide a convenient means to isolatethe protein.

The chimeric DNA constructs of the present invention can furthercomprise a termination (or 3′ untranslated) region. A termination regionrefers to that portion of a gene comprising a DNA segment that containsa polyadenylation signal and any other regulatory signals capable ofeffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by effecting the addition of polyadenylic acidtracks to the 3′ end of the mRNA precursor. Polyadenylation signals arecommonly recognized by the presence of homology to the canonical form5-AATAAA-3 although variations are not uncommon.

Examples of suitable termination regions are the 3′ transcribednon-translated regions containing a polyadenylation signal ofAgrobacterium tumour inducing (Ti) plasmid genes, such as the nopalinesynthase (Nos gene) and plant genes such as the soybean storage proteingenes and the small subunit of the ribulose-1,5-bisphosphate carboxylase(ssRUBISCO) gene.

The termination region operably linked to the heterologous gene will beprimarily one of convenience, since in many cases termination regionsappear to be relatively interchangeable.

The DNA constructs of the present invention can also include furtherenhancers, either translation or transcription enhancers, as may berequired. These enhancer regions are well known to persons skilled inthe art, and can include the ATG initiation codon and adjacentsequences. The initiation codon must be in phase with the reading frameof the coding sequence to ensure translation of the entire sequence. Thetranslation control signals and initiation codons can be from a varietyof origins, both natural and synthetic. Translational initiation regionsmay be provided from the source of the transcriptional initiationregion, or from the structural gene. The sequence can also be derivedfrom the promoter selected to express the gene, and can be specificallymodified so as to increase translation of the mRNA.

The DNA constructs of the present invention can further comprise signalpeptides operably linked to a gene of interest such that expression istargeted to a specific organelle.

A variety of techniques are available for the introduction of DNA intohost cells. For example, the chimeric DNA constructs may be introducedinto host cells using standard Agrobacterium vectors by transformationprotocols (EP 131320 B1, U.S. Pat. No. 5,591,616, U.S. Pat. No.5,149,645, U.S. Pat. No. 4,693,976; all of which are incorporated hereinby refernece). The use of T-DNA for transformation of plant cells hasreceived extensive study and is amply described in EP 120516 (also seeHoekema et al., 1985, Chapter V, In: The Binary Plant Vector SystemOffset-drukkerij Kanters B. V., Alblasserdam; Knauf, et al., 1983,Genetic Analysis of Host Range Expression by Agrobacterium, p. 245, In:Molecular Genetics of the Bacteria-Plant Interaction, Puhler, A. ed.,Springer-Verlag, NY; and An et. al., 1985, EMBO J., 4:277-284, which areincorporated herein by reference).

The use of non-Agrobacterium techniques permits the use of theconstructs described herein to obtain transformation and expression in awide variety of monocotyledonous and dicotyledonous plants and otherorganisms. These techniques include biolistics (U.S. 5,865,796, U.S.Pat. No. 5,120,657, U.S. Pat. No. 5,371,015, U.S. Pat. No. 5,179,022;which are incorporated herein by reference), electroporation (U.S.5,859,327, U.S. Pat. No. 6,002,070; Fromm et al., 1985, Proc. Natl.Acad. Sci. USA, 82:5824-5828; Riggs and Bates, 1986, Proc. Natl. Acad.Sci. USA 83:5602-5606; which are incorporated herein by reference),microinjection of protoplasts, (U.S. Pat. No. 4,743,548, which isincorporated herein by reference), penetration of cells with tungstenwhiskers, (U.S. Pat. No. 5,302,523, U.S. Pat. No. 5,464,765, which areincorporated herein by reference), lasers, (U.S. Pat. No. 5,013,660,which is incorporated herein by reference), sonification, (U.S. Pat. No.5,693,512, which is incorporated herein by reference) or PEG-mediatedDNA uptake (Potrykus et al., 1985; Mol. Gen. Genet., 199:169-177; U.S.Pat. No. 5,453,367, which are incorporated herein by reference).

The expression cassette may be joined to a marker for selection in plantcells. Conveniently, the marker may be resistance to a herbicide, egphosphinthricin or glyphosate, (U.S. Pat. No. 5,4553,367, U.S. Pat. No.4,940,835, U.S. Pat. No. 5,648,477) or an antibiotic, such as kanamycin,6418, bleomycin, hygromycin, chloramphenicol, (for example U.S. Pat. No.5,116,750, U.S. Pat. No. 6,048,730) or the like. Similarly, enzymesproviding for production of a compound identifiable by colour changesuch as GUS (β□-glucuronidase), or luminescence, such as luciferase orGFP are also useful.

Also considered part of this invention are transgenic plants containingthe gene construct of the present invention. Methods of regeneratingwhole plants from plant cells are known in the art, and the method ofobtaining transformed and regenerated plants is not critical to thisinvention. In general, transformed plant cells are cultured in anappropriate medium, which may contain selective agents such asantibiotics, where selectable markers are used to facilitateidentification of transformed plant cells. Once callus forms, shootformation can be encouraged by employing the appropriate plant hormonesin accordance with known methods and the shoots transferred to rootingmedium for regeneration of plants. The plants may then be used toestablish repetitive generations, either from seeds or using vegetativepropagation techniques.

Plants thus obtained may be cultivated and used for the production ofvarious proteins. It is envisioned that for some applications theharvested material will be subject to purification and the heterologousprotein isolated in a substantially pure form. In other instances theharvested plant material will be used as edible or oral-vaccines ortherapeutic agents. In addition, the foreign protein of interest may bepurified from the harvested plant material and may be formulated into aform for oral use or an injectable dosage form. In still other examplesthe harvested plant material may be used directly in an industrialprocess. Thus, the isolation of harvest inducible DNA sequences allowfor many strategies for the production of heterologous proteins.

The above description is not intended to limit the claimed invention inany manner, furthermore, the discussed combination of features might notbe absolutely necessary for the inventive solution.

The present invention will be further illustrated in the followingexamples. However it is to be understood that these examples are forillustrative purposes only, and should not be used to limit the scope ofthe present invention in any manner.

EXAMPLES Example 1 Isolation of Harvest-Inducible (HI) cDNA Clones

HI cDNAs were isolated from a cDNA subtractive library, made from mRNAobtained from field harvested alfalfa, as shown in FIG. 1, using aPCR-Select™ kit from ClonTech (Protocol #Pt1117-1, www.clontech.com).Briefly, this technique compares two populations of mRNA and obtainsclones of genes that are expressed in one population but not in theother.

First, two mRNA populations were converted into cDNA: the cDNA thatcontained the harvest-specific transcripts, referred to as the “tester”cDNA and the reference cDNA from the non-harvested plants, referred toas “driver” cDNA. The tester and driver cDNAs were digested with Rsa I(a four-base-cutting restriction enzyme that yields blunt ends). Thetester cDNA was subdivided into two portions, and each ligated with adifferent cDNA adaptor. The ends of the adaptor do not have a phosphategroup, so only one strand of each adaptor attaches to the 5′ ends of thecDNA. The two adaptors have stretches of identical sequence to allowannealing of the PCR primer once the recessed ends have been filled in(See FIG. 2).

Two hybridizations were then performed. In the first, an excess ofdriver was added to each sample of tester. The samples were then heatdenatured and allowed to anneal, generating the type a, b, c, and dmolecules in each sample (see FIG. 1). The concentration of high- andlow-abundance sequences is thought to be equalized among the type amolecules because reannealing is faster for the more abundant moleculesdue to the second-order kinetics of hybridization. At the same time, thesingle stranded (ss) type a molecules are significantly enriched fordifferentially expressed sequences, as cDNAs that are not differentiallyexpressed form type c molecules with the driver.

During the second hybridization, the two primary hybridization sampleswere mixed together without denaturing. As a result, only the remainingequalized and subtracted ss tester cDNAs could reassociate and form newtype e hybrids. These new hybrids are double stranded (ds) testermolecules with different ends, which correspond to the sequences ofadaptors 1 and 2R (FIG. 2). Fresh denatured driver cDNA was added,without denaturing the subtraction mix, to further enrich fraction e fordifferentially expressed sequences. After filling in the ends by DNApolymerase, the type e molecules—the differentially expressed(harvest-inducible) tester sequences—have different annealing sites forthe nested primers on their 5′ and 3′ ends.

The entire population of molecules was then subjected to PCR to amplifythe harvest-inducible sequences. During PCR, type a and d molecules aremissing primer annealing sites, and thus cannot be amplified. Due to thesuppression PCR effect, most type b molecules form a pan-like structurethat prevents their exponential amplification (see Suppression-PCReffect, below). Type e molecules have only one primer annealing site andcan only be amplified linearly. Only type e molecules, which have twodifferent adaptors, can be amplified exponentially. These are theequalized, differentially expressed sequences specific to harvestedtissue.

Next, a secondary PCR amplification was performed using nested primersto further reduce any background PCR products and to enrich forharvest-specifc sequences. The cDNAs were then directly inserted intoTopo™, a T/A cloning vector from Invitrogen.

Suppression-PCR

The PCR-Select cDNA adaptors are engineered to prevent undesirableamplification during PCR using suppression PCR (U.S. Pat. No.5,565,340). Suppression occurs when complementary sequences are presenton each end of a ss cDNA. During each primer annealing step, thehybridization kinetics strongly favor (over annealing of the shorterprimers) the formation of a pan-like secondary structure that preventsprimer annealing. When occasionally a primer anneals and is extended,the newly synthesized strand will also have the inverted terminalrepeats and form another pan-like structure. Thus, during PCR,nonspecific amplification is efficiently suppressed, and specificamplification of cDNA molecules with different adaptors at both ends canproceed normally. The 5′ ends of Adaptors 1 and 2R have an identicalstretch of 22 nucleotides (FIG. 2). Primary PCR therefore requires onlyone primer for amplification, eliminating the problem of primerdimerization. Furthermore, the identical sequences on the 3′ and 5′ endsof the differentially expressed molecules introduce a slight suppressionPCR effect. Since these identical sequences are the same length as PCRPrimer 1, the suppression effect becomes significant only for very shortcDNAs (under 200 nt), because the formation of pan structures forshorter molecules is more efficient. Thus, longer molecules arepreferentially enriched. This enrichment for longer molecules balancesthe inherent tendency of the subtraction procedure to favor short cDNAfragments, which are more efficiently hybridized, amplified, and clonedthan longer fragments.

Plant Material

The field of alfalfa (c.v. Gala, Northrup King), located on the southedge of Guelph, was in its second year after planting, and had alreadyundergone its first harvest of the season. Plants at the bud stage andready for the second harvest were cut approximately 8 cm from the basefrom a 1.0 m² area The temperature in the field was approximately 25-28°C., and the harvesting was performed at noontime to avoid humidity. Thecontrol, non-harvest-treatment plant tissue was immediately frozen inliquid nitrogen. The harvest-treatment sample was laid on the ground ina swath to wilt, as is done during conventional harvesting of this crop.After one hour, the harvested plant tissue was brought back to the lab,wrapped in tinfoil and left at ambient temperature (20° C.) on thebench.

The leaves were collected for the analysis at different harvest times—30minutes, 45 minutes, 2 hours, 6 hours, 24 hours. Total RNA was isolatedfrom both non-harvested and harvested plant materials. cDNA wasgenerated from both tissues with HI samples designated as the testerpopulation and the non-harvested samples were designated as the driverpopulation. Harvest-inducible cDNAs were inserted into TOP02.1 vector(Invitrogen).

Twelve cDNA clones, ranging in size from 180 to 500 bp, were obtainedusing the subtractive protocol outlined above. These clones weresequenced to determine redundancy and to select candidates for furtheranalysis. Seven clones of the 12 were independent and 4 (H1, H7, H11,and H12) were selected for Northern analysis. Of these, H7 (SEQ IDNO:1), H11 (SEQ ID NO:2) and H12 (SEQ ID NO:3) showed substantial andlengthy (>24 h) up-regulation following harvest and no transcription innon-harvested plants (see Northern blots, Example 2)

The DNA sequence of selected clones was determined and GeneBank searchesperformed using BLAST searching algorithm (default parameters).

Isolation of Complete cDNA Clones

To isolate the regulatory regions of H7 and H11 the complete codingregion of these genes was identified. This was done by extending ourcandidate cDNA clones in both the 5′ and 3′ directions using the RACE(rapid amplification of cDNA ends) method.

Specifically, a cDNA population was generated from alfalfa leaves (c.v.Gala) grown in a greenhouse 12 hours after harvesting using the SMART™RACE cDNA Amplification Kit from ClonTech according to manufacturer'sinstructions. Harvested plants were wilted for one hour in thegreenhouse followed by wrapping in tin foil and incubation on the labbench at 20° C. By this method of repeated “cDNA walking” and isolationof many cDNA clones overlapping each other and the original cDNA cloneisolated by subtraction, the full-length transcripts were accuratelydetermined for H7 (SEQ ID NO:1) and H11 (SEQ ID NO:2). The regulatoryregions flanking the coding region were then isolated by genomic walking(see below).

As H12 (SEQ ID NO:3) was virtually identical to an alfalfa cDNA alreadycharacterized and resident in GenBank, we did not extend H12 by RACE,but rather performed genomic walking upstream of the 5′ end of the cDNAbased on the sequence data available. The sequence of H12 shows homologyto a cDNA from alfalfa that presumably encodes the enzyme CcoMT, thoughtto be involved in lignin formation (see FIG. 7 for sequence comparison).

Example 2 Analysis of Expression Patterns of Harvest Inducible (II)cDNAs

Northern blots were done using standard protocols (Sambrook et al, 1989,in “Molecular cloning: a laboratory manual”, 2^(nd) edition, Cold SpringHarbor, N.Y. Cold Spring Harbor Laboratory). Equivalent amounts of totalRNA from harvest-induced, heat-shock treated and wounded leaf tissuewere used for hybridization. The hybridizations were overnight (12-24hrs) at 42° C. with ³²P-labelled HI cDNA presence of 50% formamide,followed by washing using standard protocols (Sambrook et al, 1989). Awounding treatment was applied to alfalfa plants by lightly scoring aleaf with a surgical blade on the leaf surface. The wounded leaves wereremoved from the plants for analyses 30 minutes, 6 hour and 24 hourspost treatment. Heat-shock treatments were performed by placing pottedalfalfa plants into an oven for 15 minutes or 30 minutes at 38° C. Thetissue samples were collected from the plants immediately following heattreatment. mRNA accumulation for cDNA clones H7, H11 and H12 wereexamined under harvest conditions. The Northern analysis results showedsignificant mRNA accumulation following harvesting but not wounding(Table 1, FIGS. 3-6). TABLE 1 Relative accumulation of HI cDNAsfollowing harvesting, wounding and heat shock treatments compared withuntreated tissue. Relative transcript level under different treatmentscDNA clones harvest heat shock wounding H1 +* ? ? H7 ++ − − H11 ++ +++ ?H12 ++ − ?*“+” and “++” are results compared with control sample where control isconsider as “−”.

Northern analysis of H7 (SEQ ID NO:1) expression before or after harvestis shown in FIG. 3. H7 expression increases following harvest, however,no expression is observed pre-harvest, or following wounding or heatshock treatments (data not shown).

Expression of H11 (SEQ ID NO:2) following harvesting or heat shock isshown in FIG. 4. Increased expression is observed following harvestingor heat shock treatment. Similarly, increased expression of H11 isobserved following wounding (FIG. 5).

H12 (SEQ ID NO:3) expression is shown in FIG. 6, where an increase inexpression is observed following harvest of plant material. Noexpression is observed in pre-harvested tissue. A low level ofexpression is detected in response to a heat shock treatment.

Example 3 Isolation of Genomic Sequences and Promoter Regions of HIGenes

Alfalfa leaf tissue was collected from plants grown in the greenhouse.Genomic DNA was isolated using a method modified from Davies (Davies LG, Dibner M D, Batty J F: Basic methods in molecular biology. Elsevier,N.Y. 1986, which is incorporated herein by reference). Construction ofthe genomic walking. “library” was performed according to themanufacturer's manual (GenomeWalker™ Kits CLONTECH, USA PT116-1). DNAfrom colonies was sequenced to find those containing inserts overlappingthe cDNA-labelled cDNA clones H7, H11 and H12 were used for screening ofthe library.

As a result of this screening, corresponding genomic DNAs, of H7 (SEQ IDNO:7, FIG. 8), H11 (SEQ ID NO:8, FIG. 9), and H12 (SEQ ID NO:9, FIG. 10)were obtained. Further analysis of these genomic DNA's was carried outto identify the associated regulatory regions of H7 (SEQ ID NO:4), H11(SEQ ID NO:5) and H12 (SEQ ID NO:6).

The regulatory regions of these genes may be used to drive theexpression of a coding sequence of interest, for example, but notlimited to the coding sequence of interest as shown in FIG. 7.

Example 4 Transgenic Plants Expressing Harvest-Inducible Promoters

Vector Construction

In order to test expression of transgenes controlled by the harvestinducible (HI) promoters isolated from alfalfa, the HI promoters werefused to the beta-glucuronidase (GUS) reporter gene and histochemicalassays conducted for GUS gene activity, which results in a blue colourin plant tissue (Jefferson et al., 1987, EMBO J. 6:3901-7). The putativeHI promoter sequences were sub-cloned from Topo (InVitrogen) orpBluescript vectors using conventional molecular techniques and existingrestriction sites or sites created by polymerase chain reaction (PCR).

The putative promoter region for the H7 cDNA clone was fused to the 5′terminus of the GUS gene in the vector pBI101 (Jefferson et al., 1987,EMBO J. 6:3901-7, FIG. 1 a), using HindIII and Xba1; in addition, the H7promoter was fused to the GUS gene in pCAMBIA3301 (CAMBIA), using KpnIand Xba1 (FIG. 11B). The H11 promoter was fused to pCAMBIA2301, and theH12 promoter was fused to pCAMBIA1303 (FIGS. 11C, D). In all cases, thepromoter is also 5′ to the GUS gene.

All of the above vectors are of the binary type, which means they can begrown in both E. coli and Agrobacterium, the latter for transfer of theregions between the left and right borders to the plant genome.

Transfer of HI-GUS Constructs to Plants

The binary vectors were transferred to Agrobacterium tumefaciens strainC58 (Rif res) containing the helper plasmid pMP90. The procedure forcocultivation of sterile leaves from 4-week old tobacco plants (cultivarPetH4) and regeneration followed the method of Fisher and Guiltinan(Fisher & Guiltinan, 1995, Plant Mol Biol Rep. 13:278-89). Selection oftransgenic tissue and shoots was facilitated by incorporation ofkanamycin (300 mg/l) or hygromycin (25 mg/l), depending on the vectorused (see FIG. 11).

The binary vectors were also used to transfer HI-GUS constructs toMedicago truncatula according to the seedling infiltration method ofTrieu et al. (Trieu A T, et al., 2000, Plant J. 22:531-41).

Histochemical GUS Assays for Transgene Expression

Tobacco leaves from regenerated plants grown in the greenhouse, andleaves and stems from M. truncatula plants grown from the cocultivatedseedlings were incubated in the X-gluc substrate and the green pigmentsremoved for visualization of the blue precipitate resulting from GUSenzyme activity (Jefferson et al., 1987, EMBO J. 6:3901-7).

Results

Analysis of tobacco R0 (primary) regenerants, 5-10 plants for each ofthe above constructs, showed GUS gene expression (i.e. blue colouration)24 hrs following harvesting whereas none was evident in plants at timezero or in the non-transgenic controls (FIG. 12). Random sampling ofportions (leaves and stem/petiole sections) of the M. trunculata plantsthat had undergone cocultivation at the seedling stage also revealeddistinct blue colouration in some sectors, but not in all parts and onlyafter the harvesting treatment (FIG. 13). The sectoral pattern of theblue stain reflects the chimeric nature of gene transfer, and thecocultivation of intact seedlings. Once again, no blue colour wasevident in the transgenic plants at time zero or in the non-transgeniccontrols. It is also significant that the extent of blue colouration wasgreater in the case of constructs containing the H11 and H7 promotersthan in plants containing the conventional 35S promoter. The lack ofblue colour in transgenic trunculata plants at time zero demonstratesthat the blue colour was not due to endogenous GUS activity in residualAgrobacteria.

The extent and intensity of blue colouration in the HI-GUS plants of thepresent invention noticeably exceeds that of plants containing the GUSgene controlled by the 35S promoter. The latter promoter is derived fromthe cauliflower mosaic virus and is considered to be a constitutivepromoter, which provides a high level of expression to transgenes,especially in tobacco. Therefore, not only do the HI promoters of thepresent invention avoid the problems associated with constitutiveexpression, but they also exceed the levels of expression provided byone of the strongest constitutive promoter available for plants.

As presently shown, the expression of the HI promoters is tightlyregulated in that repeatedly no expression has been observed in thetransgenic plants of the present invention at time zero, and does notappear until several hours after harvesting. It is also significant thatno additional wounding of the plant tissue is needed to obtain highexpression levels throughout all harvested tissue, although additionalwounding or other treatments such as heat may augment expressions levelseven further.

Furthermore, the HI-GUS transgenes show the same harvest-specificinduction patterns in tobacco and M truncatula as do the native cDNAclones in alfalfa from which they were isolated under harvestingconditions. Although M trunculata is a close relative of alfalfa,tobacco is quite distant phylogenetically from alfalfa. This shows thatthe HI promoters of the present invention are regulated in a similarpattern in other plant families and genera, such as the grass speciesand have applications in crops of such species.

The following table (Table 2) is a summary of the Sequence ID numbersdefined in the present application. TABLE 2 Sequence ID numbers definedin the present invention. Sequence ID No. Description Figure SEQ ID NO:1 Nucleotide sequence of 8 H7 coding region SEQ ID NO: 2 Nucleotidesequence of 9 H11 coding region SEQ ID NO: 3 Nucleotide sequence of 10H12 coding region SEQ ID NO: 4 Nucleotide sequence of 8 H7 regulatoryregion SEQ ID NO: 5 Nucleotide sequence of 9 H11 regulatory region SEQID NO: 6 Nucleotide sequence of 10 H12 regulatory region SEQ ID NO: 7Nucleotide sequence of 8 genomic H7 SEQ ID NO: 8 Nucleotide sequence of9 genomic H11 SEQ ID NO: 9 Nucleotide sequence of 10 genomic H12 SEQ IDNO: 10 Amino acid sequence 8 encoded by H7 coding region SEQ ID NO: 11Amino acid sequence 10 encoded by H12 coding region SEQ ID NO: 12Nucleotide sequence of 2 PCR-Select cDNA synthesis primer SEQ ID NO: 13Nucleotide sequence of 2 Adaptor 1 SEQ ID NO: 14 Nucleotide sequence of2 Adaptor 2R SEQ ID NO: 15 Nucleotide sequence of 2 PCR primer 1 SEQ IDNO: 16 Nucleotdie sequence of 2 nested PCR primer 1 SEQ ID NO: 18Nucleotide sequence of 2 complement (partial) SEQ ID NO: 19 Nucleotidesequence of 2 complement (partial) SEQ ID NO: 17 Nucleotdie sequence of2 nested PCR primer 2RAll citations are herein incorporated by reference.

The present invention has been described with regard to preferredembodiments. However, it will be obvious to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as described herein.

1. A method for isolating a harvest-inducible DNA sequence comprising:i) constructing one, or more than one, first cDNA libraries comprisingcDNA sequences expressed in harvested tissue; ii) preparing one, or morethan one, second cDNA libraries comprising cDNA sequences expressed intissues of an intact plant prior to harvest; and iii) identifying theharvest-inducible cDNA sequence present in the one, or more than one,first cDNA library that is not present in the second cDNA library.
 2. Anisolated harvest-inducible cDNA sequence obtained according to themethod of claim
 1. 3. An isolated harvest-inducible cDNA sequenceselected from the group consisting of: i) SEQ ID NO:1, a complementthereof, a fragment of SEQ ID NO:1, a complement of a fragment of SEQ IDNO:1, a nucleic acid that hybridizes to SEQ ID NO:1 under stringenthybridization conditions, a nucleic acid that hybridizes to a complementof SEQ ID NO:1 under stringent hybridization conditions, a nucleic acidthat hybridizes to a fragment of SEQ ID NO:1 under stringenthybridization conditions, or a nucleic acid that hybridizes to acomplement of fragment of SEQ ID NO:1 under stringent hybridizationconditions; ii) SEQ ID NO:2, a complement thereof, a fragment of SEQ IDNO:2, a complement of a fragment of SEQ ID NO:2, a nucleic acid thathybridizes to SEQ ID NO:2 under stringent hybridization conditions, anucleic acid that hybridizes to a complement of SEQ ID NO:2 understringent hybridization conditions, a nucleic acid that hybridizes to afragment of SEQ ID NO:2 under stringent hybridization conditions, or anucleic acid that hybridizes to a complement of fragment of SEQ ID NO:2under stringent hybridization conditions; and iii) SEQ ID NO:3, acomplement thereof, a fragment of SEQ ID NO:3, a complement of afragment of SEQ ID NO:3, a nucleic acid that hybridizes to SEQ ID NO:3under stringent hybridization conditions, a nucleic acid that hybridizesto a complement of SEQ ID NO:3 under stringent hybridization conditions,a nucleic acid that hybridizes to a fragment of SEQ ID NO:3 understringent hybridization conditions, or a nucleic acid that hybridizes toa complement of fragment of SEQ ID NO:3 under stringent hybridizationconditions, the stringent hybridization conditions comprising,hybridization overnight (12-24 hrs) at 42° C. in the presence of 50%formamide, followed by washing, or 5×SSC at about 65° C. for about 12 toabout 24 hours, followed by washing in 0.1×SSC at 65° C. for about onehour.
 4. A method for isolating a harvest inducible regulatory elementcomprising, i) identifying genomic DNA sequences 3′ and 5′ correspondingto the harvest-inducible cDNA identified using the method of claim 1;and ii) analyzing the genomic DNA, and identifying the harvest-inducibleregulatory element.
 5. The method of claim 4 further comprising a stepof: iii) testing said harvest-inducible regulatory region within atransgenic plant or plant cell.
 6. A harvest-inducible regulatoryelement obtained using the method of claim
 4. 7. A harvest-inducibleregulatory element selected from the group consisting of: i) SEQ IDNO:4, a complement thereof, a fragment of SEQ ID NO:4, a complement of afragment of SEQ ID NO:4, a nucleic acid that hybridizes to SEQ ID NO:4under stringent hybridization conditions, a nucleic acid that hybridizesto a complement of SEQ ID NO:4 under stringent hybridization conditions,a nucleic acid that hybridizes to a fragment of SEQ ID NO:4 understringent hybridization conditions, or a nucleic acid that hybridizes toa complement of fragment of SEQ ID NO:4 under stringent hybridizationconditions; ii) SEQ ID NO:5, a complement thereof, a fragment of SEQ IDNO:5, a complement of a fragment of SEQ ID NO:5, a nucleic acid thathybridizes to SEQ ID NO:5 under stringent hybridization conditions, anucleic acid that hybridizes to a complement of SEQ ID NO:5 understringent hybridization conditions, a nucleic acid that hybridizes to afragment of SEQ ID NO:5 under stringent hybridization conditions, or anucleic acid that hybridizes to a complement of fragment of SEQ ID NO:5under stringent hybridization conditions; and iii) SEQ ID NO:6, acomplement thereof, a fragment of SEQ ID NO:6, a complement of afragment of SEQ ID NO:6, a nucleic acid that hybridizes to SEQ ID NO:6under stringent hybridization conditions, a nucleic acid that hybridizesto a complement of SEQ ID NO:6 under stringent hybridization conditions,a nucleic acid that hybridizes to a fragment of SEQ ID NO:6 understringent hybridization conditions, or a nucleic acid that hybridizes toa complement of fragment of SEQ ID NO:6 under stringent hybridizationconditions, the stringent hybridization conditions comprising,hybridization overnight (12-24 hrs) at 42° C. in the presence of 50%formamide, followed by washing, or 5×SSC at about 65° C. for about 12 toabout 24 hours, followed by washing in 0.1×SSC at 65° C. for about onehour, wherein the regulatory element exhibits harvest-inducibleactivity.
 8. A construct comprising said harvest-inducible regulatoryelement of claim 7, operably linked with a heterologous coding sequenceof interest and a terminator region.
 9. A construct comprising aheterologous coding sequence operably linked to the harvest-inducibleregulatory element of claim 7, the harvest-inducible regulatory elementfurther comprising a nucleotide sequence encoding a harvest-inducibleprotein or fragment thereof.
 10. A vector comprising the DNA constructof claim
 8. 11. A vector comprising the DNA construct of claim
 9. 12. Aplant, plant tissue, plant seed, plant cell, or progeny therefrom,comprising the construct of claim
 8. 13. A plant, plant tissue, plantseed, plant cell, or progeny therefrom, comprising the construct ofclaim
 9. 14. A method for production of a heterologous protein in aplant comprising: i) providing a plant transformed with the construct ofclaim 8; ii) growing the transformed plant; and iii) harvesting thetransformed plant thereby inducing expression of the heterologousprotein.
 15. The method of claim 14, wherein the step of harvesting(step iii) is followed by: iv) isolating the heterologous protein fromthe transformed plant.
 16. The method of claim 15, wherein the step ofisolating (step iv)) is followed by a step of purification of theheterologous protein.
 17. A method for production of a heterologousprotein in a plant comprising, i) providing a plant transformed with theconstruct of claim 9; ii) growing the transformed plant; and iii)harvesting the transformed plant to induce expression of theheterologous protein.
 18. The method of claim 17, wherein the step ofharvesting (step iii) is followed by: iv) isolating the heterologousprotein from the transformed plant.
 19. The method of claim 18, whereinthe step of isolating (step iv)) is followed by a step of purificationof the heterologous protein.
 20. A method for production of aheterologous protein in a plant comprising: i) providing a plantexpressing the construct of claim 8; ii) growing the plant; and iii)harvesting the plant thereby inducing expression of the heterologousprotein.
 21. A method for production of a heterologous protein in aplant comprising, i) providing plant expressing the construct of claim9; ii) growing transformed plant; and iii) harvesting the plant toinduce expression of the heterologous protein.
 22. A harvest inducibleregulatory element according to claim 7, wherein the harvest inducibleregulatory element is SEQ ID NO:4.
 23. A harvest inducible regulatoryelement according to claim 7, wherein the harvest inducible regulatoryelement is SEQ ID NO:5.
 24. A harvest inducible regulatory elementaccording to claim 7, wherein the harvest inducible regulatory elementis SEQ ID NO:6.
 25. The plant, plant tissue, plant seed, plant cell, orprogeny therefrom according to claim 12, wherein the plant, planttissue, plant seed, plant cell, or progeny therefrom is selected fromthe group consisting of potato, tomato, canola, corn, soybean, alfalfa,pea, lentil, other forage legumes such as clover, trefoil, foragegrasses such as timothy, ryegrass, brome grass, fescue or other cerealgrasses used for forage such as barley, wheat, sudan grass, sorgham. 26.The plant, plant tissue, plant seed, plant cell, or progeny therefromaccording to claim 13, wherein the plant, plant tissue, plant seed,plant cell, or progeny therefrom is selected from the group consistingof potato, tomato, canola, corn, soybean, alfalfa, pea, lentil, otherforage legumes such as clover, trefoil, forage grasses such as timothy,ryegrass, brome grass, fescue or other cereal grasses used for foragesuch as barley, wheat, sudan grass, sorgham.